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Inversed PCR issues


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#1 Ravafa

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Posted 19 August 2009 - 02:51 AM

Hi there!

I am trying to run inversed PCR and i am unsuccessful so far. I have primers designed inside my region that i would like to amplify - both ways: going in as a positive control and going out to amplify flanking region.

I will briefly describe my experiment:

1) DNA isolation: using combined method of CTAB and DNeasy Kit. I am diluting in water to avoid EDTA inhibition. Checked on nanodrop, DNA yield is between 50 and 100 ng/ul.
- PCR with control primers is working

2) Digestion: with EcorI and MboI separately. 30 U for 18 hours with 1-3 ug of genomic DNA in total volume of 30ul, 37 C, in thermalcycler. I repeat this step for better digestion and than i leave it for 20 minutes in 65 C to disactivate enzymes.
- there is a smear on the gel
- control: i had 450bp amplicon with MboI site, I used 1ug of this and all was digested into 250 and 200 bp fragments.
- PCR with control primers is working

3) Ligation: with T4 ligase, 18h in 16 C in thermal cycler,
- I tried both: 20 ul volume (15 ul of DNA - 0,5 ug; 4 ul of buffer 5x; 1U of ligase) and 100 ul (15 ul of DNA; 20 ul of buffer; 3U of ligase; 65 ul H2O as advised in one paper)
- control:digested amplicon: approximately half of the DNA was ligated back (visible bends: 200bp, 250bp, 450bp, 400bp and 500bp)

4) Purification: Ethanol precipitation.
-0,2 volumes of 5 M NaCl, 3 vol. of Ethanol 99%, kept in fridge for 2 hours
- centrifugation at 14000 for 30 min, remove supernatant
- rinced with 1 vol. 75% Ethanol
- centrifugation for 14000 for 10 min, remove supernatant
- diluting pelet in 20 ul of water for 1 h RT

- on nanodrop i get 150-1500 ng/ul of DNA

5) PCR: 30 cycles: 94 - 30s; 52 - 30s; 72 - 2 min, separatly with both primers
- no product
- control: no result in here neither.

So clearly I have PCR inhibition, but why is that?

If you have any ideas, please help me.

Bests,

#2 phage434

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Posted 21 August 2009 - 06:20 PM

It's likely to be too much DNA in both the ligation and pcr reactions. You want to circularize the DNA fragments after digestion, which is the only way to create molecules which will act as PCR templates. Circularization competes with ligation to random other cut ends. You want the local concentration of the other end of a cut fragment to be higher than the local concentration of other cohesive ends that can ligate. This can only occur in dilute solutions. The cut DNA you have is likely perfectly fine, although I don't think you need to cut it as long or twice, as you have done. An hour at 37 should be more than enough, followed by heat killing. I would not bother to purify the DNA after digestion -- it will just cause problems. Use about 5 ng of cut DNA in your ligation reaction, which can be in very low volume. Heat kill the ligase, and add 1 ul of the ligation reaction directly to a PCR reaction with your outwardly directed primers. Less is more. Run the PCR for 35 cycles or more.

It would be best to test your primers. If you can do a PCR reaction with each of these primers as half of a pair, that would be ideal, since you don't know if your primers are working well (or at all). What's the organism GC content -- you may be having PCR problems from the DNA sequence.

If you are using 5x ligation buffer, then you are probably doing a "quick ligation" kit ligation, which I would not recommend. Switch to normal ligase buffer for all cohesive end ligations.




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